RESEARCH ARTICLE

Complement Component C3 andButyrylcholinesterase Activity Are Associatedwith Neurodegeneration and ClinicalDisability in Multiple SclerosisShahin Aeinehband1☯*, Rickard P. F. Lindblom1☯, Faiez Al Nimer1,Swetha Vijayaraghavan2, Kerstin Sandholm4, Mohsen Khademi1, Tomas Olsson1,Bo Nilsson3, Kristina Nilsson Ekdahl3,4, Taher Darreh-Shori2, Fredrik Piehl1

1 Department of Clinical Neuroscience, Neuroimmunology Unit, Karolinska Institutet, Stockholm, Sweden,2 Division of Alzheimer Neurobiology Center, Department of Neurobiology, Care Sciences and Society,Karolinska Institutet, Stockholm, Sweden, 3 Division of Clinical Immunology, Department of Immunology,Genetics and Pathology, Uppsala University, Uppsala, Sweden, 4 School of Natural Sciences, LinnæusUniversity, Kalmar, Sweden

☯ These authors contributed equally to this work.* shahin.aeinehband@ki.se

AbstractDysregulation of the complement system is evident in many CNS diseases but mechanisms

regulating complement activation in the CNS remain unclear. In a recent large rat genome-

wide expression profiling and linkage analysis we found co-regulation of complement C3

immediately downstream of butyrylcholinesterase (BuChE), an enzyme hydrolyzing acetyl-

choline (ACh), a classical neurotransmitter with immunoregulatory effects. We here deter-

mined levels of neurofilament-light (NFL), a marker for ongoing nerve injury, C3 and activity

of the two main ACh hydrolyzing enzymes, acetylcholinesterase (AChE) and BuChE, in ce-

rebrospinal fluid (CSF) from patients with MS (n = 48) and non-inflammatory controls (n =

18). C3 levels were elevated in MS patients compared to controls and correlated both to dis-

ability and NFL. C3 levels were not induced by relapses, but were increased in patients with

�9 cerebral lesions on magnetic resonance imaging and in patients with progressive dis-

ease. BuChE activity did not differ at the group level, but was correlated to both C3 and NFL

levels in individual samples. In conclusion, we show that CSF C3 correlates both to a mark-

er for ongoing nerve injury and degree of disease disability. Moreover, our results also sug-

gest a potential link between intrathecal cholinergic activity and complement activation.

These results motivate further efforts directed at elucidating the regulation and effector func-

tions of the complement system in MS, and its relation to cholinergic tone.

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OPEN ACCESS

Citation: Aeinehband S, Lindblom RPF, Al Nimer F,Vijayaraghavan S, Sandholm K, Khademi M, et al.(2015) Complement Component C3 andButyrylcholinesterase Activity Are Associated withNeurodegeneration and Clinical Disability in MultipleSclerosis. PLoS ONE 10(4): e0122048. doi:10.1371/journal.pone.0122048

Academic Editor: Israel Silman, Weizmann Instituteof Science, ISRAEL

Received: September 12, 2014

Accepted: February 6, 2015

Published: April 2, 2015

Copyright: © 2015 Aeinehband et al. This is an openaccess article distributed under the terms of theCreative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in anymedium, provided the original author and source arecredited.

Data Availability Statement: All relevant data arewithin the paper.

Funding: The authors have no support or funding toreport.

Competing Interests: The authors have declaredthat no competing interests exist.

IntroductionMultiple Sclerosis (MS) is primarily an autoimmune disease of the central nervous system(CNS) characterized by chronic inflammation leading to demyelination, axonal damage andsubsequent neurological deficits [1]. With time a majority of patients with relapsing remittingMS (RRMS) will convert to a secondary progressive (SPMS) disease course [1]. A minority ofpatients displays a progressive course already from disease onset; primary progressive MS(PPMS), which has been suggested to represent RRMS “amputated” from its relapsing-remit-ting phase [1, 2].

Arguably, absence of remission in late stage RRMS reflects failed recovery processes due torepeated attacks of inflammation leading to irreversible axonal damage, the main determinantof irreversible disability in MS patients [3]. Thus, the transition to SPMS occurs when the CNScan no longer compensate for the neuronal loss [4]. However, it may also be that worsening inrelative absence of relapses and radiological signs of inflammation in progressive MS could in-volve other disease mechanisms than in the relapsing-remitting phase, which is characterizedby a T cell dependent immune response [5–7]. The complement system, an important part ofthe innate immune system with a wide range of effects in multiple disease states in the CNScould play such a contributory role [8]. In fact, several recent studies suggest that certain com-plement proteins are dysregulated in MS. For instance, certain types of MS lesions displaysprominent complement activation [9]. Moreover, elevated levels of plasma C3, C4, C4a, C1 in-hibitor, factor H, and reduced levels of C9 have been found in MS patients [10–13], as well asincreased cerebrospinal fluid (CSF) C4b and alterations in C3 [14–16].

CSF levels of the central complement component C3 have been shown to correlate with de-gree of neurological impairment in several neurodegenerative diseases [17], but any relation todisease severity in MS is unknown.

We have previously found that genetically determined susceptibility to neurodegenerationafter a standardized nerve injury among inbred rat strains is associated with a more prominentlocal inflammatory response with elevated expression of the upstream complement proteinsC1q and C3 [18]. By performing genome-wide expression profiling and linkage analysis in alarge inbred rat strain intercross we found co-regulation of complement C3 immediatelydownstream of butyrylcholinesterase (BuChE), an enzyme that hydrolyses acetylcholine (ACh)[19]. BuChE has been proposed as a risk gene for sporadic Alzheimer´s disease (AD) [20] andwe recently showed that such patients carrying a BuChE allele coding for a protein with re-duced activity displays lower levels of glial activation markers [21]. In an early study, the activi-ty of the two main ACh hydrolyzing enzymes, acetylcholinesterase (AChE) and BuChE, weredetermined in CSF and brain tissue in a small group of patients, including one MS patient thatdisplayed reduced AChE, but not BuChE activity [22]. Neuropathological studies on MS braintissue have found increased BuChE activity associated with microglia within MS lesions, whichcould contribute to the pro-inflammatory milieu [23], as well as an altered balance of cholineacetyltransferase (ChAT), the ACh synthesizing enzyme, and AChE in the hippocampus [24].However, in a recent study the activity of AChE in CSF was found not to differ between MS pa-tients and controls [25].

The aim of this study was to determine and correlate CSF levels of C3 in MS patients andcontrols, to different disease measures, including a biomarker for nerve injury, as well as to ac-tivity of the two main ACh hydrolyzing enzymes.

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Material and Methods

Clinical materialCSF samples were obtained during diagnostic workup or for diagnostic or other clinical pur-poses from patients attending the Neurology Clinic, Karolinska University Hospital, Solna.Written informed consent was obtained from all patients and the study was approved by theRegional Ethical Vetting Board of Stockholm (Diary Number: 2009/2107-31-2). Clinical exam-inations were performed by specialists in neurology, and all patients diagnosed with MS ful-filled the McDonald criteria [26]. All MS patients were evaluated clinically at time of samplingwith the Expanded Disability Status Scale (EDSS) and the subsequent calculation of MultipleSclerosis Severity Scale (MSSS). Two patients in the RRMS relapse group received disease mod-ifying treatments at time of sampling, one interferon-β1a and one natalizumab (with 8 weekswash out). Sixteen RRMS patients were in relapse defined as an increase of�1 EDSS pointwith duration of at least one week, not longer than 3 months before sampling, where systemicinfection had been ruled out. The remaining RRMS patients were in remission, defined as a sta-ble clinical and neuroradiological status�3 months prior to sampling. SPMS was defined as aninitial relapsing-remitting disease course followed by>12 months of continuous worsening ofneurological function (�0.5 EDSS point) not explained by relapses. The control group con-sisted of patients with non-inflammatory neurological/psychiatric conditions (OND) with nor-mal magnetic resonance imaging (MRI) scans and lacking signs of inflammatory activity inCSF in terms of pleocytosis or intrathecal IgG production; psychosis n = 6; functional paresthe-sia n = 4; peripheral mononeuropathy n = 3; lumbago = 1; epilepsy n = 1; vertigo n = 1; fatiguen = 1; syringomyelia n = 1). Two patients had received corticosteroids for their MS relapse; twomonths before and one week before sampling, respectively. The exclusion of the treated indi-viduals did not significantly alter the results. For patient characteristics see Tables 1 and 2. CSFcell counts, immunoglobulin content and electrophoresis were performed at the time of sam-pling at the Department of Clinical Chemistry, Karolinska University Hospital according toclinical routines.

Quantification of protein and cholinesterase enzymatic activityComplement protein C3 levels in 1:100 diluted CSF were analysed with a sandwich ELISA aspreviously described [27]. In brief, a C3 assay plate was coated with rabbit anti-C3c (A0062,Dako) diluted 1:3000. C3c recognizes C3, C3b, iC3b, and C3c. Detection of the bound C3/C3-fragments was carried out with biotinylated anti-C3c diluted 1:3200, followed by streptavi-din-HRP diluted 1:500 (Amersham). The concentration of C3, in each sample was determined

Table 1. Demographics of MS patients and controls.

Groups n Mean age (SD) Females (%) Mean EDSS (range) Mean disease duration at sampling (range)

OND 18 30.4 (7.9) 55.6 N/A N/A

RRMS 33 36.6 (9.6) 70.6 2.0 (0–4.5) 5.4 (0–19)

-Relapse 16 34.1 (8.5) 81 2.0 (0–4.5) 4.8 (0–16)

-Remission 17 39.1 (10.2) 65 1.9 (0–4) 6.0 (0.5–19)

SPMS 9 52.0 (10.1) 55.6 5.2 (4–6.5) 17 (4–25)

PPMS 6 50.3 (8.5) 33.3 4.2 (3.5–5.5) 3.8 (2–10)

All MS 48 41.2 (11.6) 64.6 3.0 (0–6.5) 7.4 (0–25)

OND = Other Neurological/psychiatric Diseases, RRMS = Relapsing-Remitting Multiple Sclerosis, SPMS = Secondary Progressive MS, PPMS = Primary

Progressive MS, EDSS = Expanded Disability Status Scale.

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using DeltaSoft (BioMetallics Inc, Princeton NJ, USA) software. A positive control of pooledplasma from five blood donors was included, and a sample with known concentrations of C3was used as a standard.

Protein levels of BuChE and AChE were quantified in CSF diluted 1:4 by using a custommade ELISA, as previously described [28].

The activities of BuChE and AChE were measured using a modified version of Ellman's col-orimetric assay, using 5.0 mM butyrylthiocholine iodide (Sigma) or 0.5 mM acetylthiocholineiodide (Sigma) as substrate in the presence of 1.0 μM of the selective AChE inhibitorBW280C51 (Sigma) or 0.1 mM of the selective BuChE inhibitor ethopropazine (Sigma) [29].Using these selective AChE and BuChE inhibitors at the specified final concentrations, a possi-ble cross contamination of the AChE and BuChE activities is expected to be less than 5%. Toeffectively minimize any intra-assay variation due to storage and sample handling, all CSF sam-ples were kept at -80C until the assay and all samples were simultaneously thawed in an ice-water bath. Samples were added in triplicates and run together on one 384-wells plate.

The MS patients included here had been genotyped with Immunochip [30], and allelic dif-ferences in the BCHE gene were determined based on the rs1803274 polymorphism.

NFL was determined with a commercial ELISA kit (UMAN Diagnostics AB) in undilutedCSF according to the manufacturer´s instructions.

All quantifications were performed blinded and patient allocation to the different groupswas performed before analyses by a single rater based on reviews of clinical records.

Statistical analysisSignificance levels were calculated using one-way ANOVA with Tukey's post-hoc tests inGraphPad Prism 5.0 (San Diego, CA). Correlations between the different protein levels was cal-culated using Pearson’s correlations assuming equal distribution depicted with simple regres-sion plots. P< 0.05 was considered statistically significant. � = p< 0.05; �� = p< 0.01; ��� =p< 0.001.

Results

Elevated CSF C3 levels in MS correlate with disease course, MRIlesions, disease severity and a biomarker of nerve injuryLevels of C3 and NFL, an established marker of neurodegeneration in MS [31–33], were ana-lyzed in CSF obtained from a cohort of patients (n = 48) with relapsing-remitting (RRMS) orprogressive (SPMS/PPMS) disease, as well as in controls with other non-inflammatory neurolog-ical/psychiatric diseases (OND; n = 18) (Fig 1A and 1B). For patient details see Tables 1 and 2.

Table 2. Characteristics of MS patients and controls.

Diagnosis n C3 median (range) μg/L NFL median (range) ng/L BuChE activity median(range) nmol/min/ml

AChE activity median(range) nmol/min/ml

CSF Cells median(range) x106/L

OND 18 1818.1 (826.9–3354) 212 (77–472.8) 6.0 (2.7–9.9) 9.8 (6.9–17.5) N/A

RRMS 33 2227.9 (856.6–4249.8) 1297.2 (293.7–9162.1) 5.7 (3.4–9.3) 8.0 (1.8–17.5) 4 (0–21)

-Relapse 16 2273.8 (1245.7–3986.6) 2237.5 (722.7–9162.1) 6.1 (4.4–9.3) 8.3 (1.8–17.5) 6 (0–21)

-Remission 17 1964.4 (856.6–4249.8) 738 (293.7–2756.1) 5.4 (3.4–8.4) 7.7 (2.8–12.1) 4 (0–21)

SPMS 9 2623.3 (1983.3–4723.3) 909.9 (280.4–4049.5) 7.0 (4.7–9.4) 5.6 (3.8–8.0) 3 (0–7)

PPMS 6 4180.8 (2279.1–5030.3) 874.5 (644.3–2940.7) 5.6 (3.7–8.4) 8.2 (3.9–12.1) 7 (0–12)

All MS 48 2400.3 (856.6–5030.3) 1028.5 (280.4–9162.1) 6.0 (3.4–9.4) 7.3 (1.8–17.5) 4 (0–21)

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Fig 1. Levels of C3 in cerebrospinal fluid are increased in patients with MS and correlate with MRI lesions, clinical disability and levels of NFL.CSFwas obtained from 48 patients diagnosed with RRMS, SPMS or PPMS and 18 controls with other neurological/psychiatric diseases (OND). Proteinquantification demonstrates increased levels of C3 in the CSF of MS patients compared to controls, with higher levels in patients with progressive disease,especially PPMS (A). Levels of NFL, a marker of neurodegeneration, are also increased in MS patients compared to controls (B). Patients with 9 or more MRIlesions at the time of sampling have significantly higher C3 levels (C) and a non-significant increase in NFL levels (D). C3 levels correlate with degree ofclinical disability as assessed with EDSS (E), and NFL (F). * = p< 0.05; ** = p< 0.01; *** = p< 0.001.

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CSF NFL levels were significantly increased in all subgroups of MS compared to OND controls,with RRMS relapse> RRMS remission> SPMS = PPMS> OND (Table 2). Also the levels ofC3 in CSF were significantly increased in MS compared to OND controls, but in a different rankorder, with PPMS> SPMS> RRMS> OND (Table 2 and Fig 1A). Separate analysis of C3 andNFL levels were done on MS patients divided after�9 (n = 40) or<9 (n = 8) cerebral MRI le-sions. MS patients that had�9 cerebral MRI lesions at the time of sampling also had significant-ly increased C3 levels compared to those with few MRI lesions (P = 0.011; Fig 1C). The sametrend was seen with NFL, however not significant (P = 0.118; Fig 1D). In the MS cohort C3 levelscorrelated with disease severity as assessed by EDSS (Fig 1E) and to a lesser degree to MSSS(p = 0.023, R2 = 0.13). The correlation between C3 and NFL in the MS cohort was not significant(p = 0.11, R2 = 0.053), but became significant when the OND group was included in the analysis(Fig 1F). In contrast, there was no correlation between C3 and disease duration (p = 0.71, R2 =0.0029), intrathecal antibody production (IgG index) (p = 0.35, R2 = 0.019) or CSF cell numbers(p = 0.81, R2 = 0.0012). There was a trend for a correlation between C3 and age in RRMS(p = 0.069, R2 = 0.1025), but not in OND (p = 0.77, R2 = 0.005).

Levels of BuChE, but not AChE correlate with CSF C3 and NFL levels inMS patientsThe genetic link between local expression of BuChE and C3 after experimental nerve injuryprovided a rationale to determine BuChE activity in the clinical samples [19,34]. In addition,activity of AChE, the other main ACh hydrolyzing enzyme [35], was determined. Enzymaticactivity and protein levels displayed a high degree of correlation for BuChE (p<0.0001,R2 = 0.887). At the group level, BuChE activity in MS patients did not differ from controls (Fig2A). However, in individual samples BuChE activity significantly correlated to both C3 andNFL in the MS group (Fig 2B and 2C), and displayed a trend for correlation to EDSS (Fig 2Cand 2D). In contrast, AChE enzymatic activity at the group level was significantly decreased inMS patients as compared to OND (Fig 2E). On an individual level AChE activity did not corre-late neither to C3 nor NFL (Fig 2F). On the contrary, there was a trend for a negative correla-tion between AChE activity and EDSS (p = 0.086, R2 = 0.064), thus, opposite to the pattern ofBuChE. MS patients carrying the BuChE-K allele displayed an 8% lower BuChE activity thannon-carriers, however, the difference was not statistically different.

RRMS clinical relapse activity increase CSF NFL levels, but not BuChEactivity or C3In the RRMS group 16 patients had a clinical relapse within 3 months of sampling, while 17were in remission. CSF levels of C3 were not different between the two groups (Fig 3). In con-trast, and as expected, NFL levels were significantly increased in patients with a recent relapse.BuChE activity displayed a non-significant trend (p = 0.061) for an increase in patients with arelapse as compared to patients in remission (Fig 3).

DiscussionWe here find increased levels of the key complement component C3 in the CSF of MS patients,which correlate with the degree of clinical disability, as well as one of the most establishedmarkers of ongoing neurodegeneration, NFL [31–33].

The MS cohort consisted mainly of untreated RRMS patients with active disease and 16 pa-tients were in relapse as defined by a worsening of neurological functions at the time of sam-pling. The group of RRMS patients was either newly diagnosed or had interrupted treatment

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due to pregnancy or personal reasons. Only two were on treatment, both RRMS with relapse;one with interferons and the other natalizumab. The levels of NFL we find here correspondwell to those we reported earlier at baseline for patients starting natalizumab treatment, withmean NFL levels at 860 ng/L for patients with no relapse activity within 3 months vs 2300 ng/Lin patients with a relapse [36]. The corresponding values here were 1129 ± 197 ng/L (n = 17)for patients in remission vs. 2965 ± 576 ng/L (n = 16) for patients with a recent relapse. Gener-ally higher levels of NFL in the RRMS group are also consistent with previous data from ourgroup in a very large cohort of MS patients, showing a negative correlation with age [37]. It ispresently unclear to what degree clinical worsening in progressive MS reflects an underlying

Fig 2. BuChE activity, but not AChE activity, correlates with levels of C3 and NFL. BuChE activity was not different between MS patients and controls atthe group level, however, with a large spread within groups (A). A highly significant correlation between BuChE activity and C3 levels is evident (B). Also,BuChE activity correlated with levels of NFL (C) and demonstrates a close to significant trend for correlation to EDSS (D). Interestingly, AChE activity differsbetween MS patients and controls (E), but lacks correlation to C3 at the individual level (F). *p =< 0.05; ** = p< 0.01; *** = p< 0.001.

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Fig 3. RRMS clinical relapse activity increase CSF NFL levels, but not BuChE activity or C3. RRMS patients in relapse (n = 16) as compared to patientsin remission (n = 17). C3 levels were not affected by clinical disease activity in RRMS (A), while NFL levels were significantly higher after relapse (B). ForBuChE activity there was a non-significant (p = 0.06) trend for an increase in patients with a relapse. * = p< 0.05; ** = p< 0.01; *** = p< 0.001.

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active inflammatory/neurodegenerative disease process [38, 39]. However, it is clear from thedata presented here that at least some patients with progressive disease display elevated NFLlevels, suggesting an active disease process. In line with this a recent study demonstrated thatimmunomodulatory treatments displayed efficacy for lowering CSF NFL levels in progressivepatients with increased baseline levels [40].

The finding of increased CSF C3 levels supports the notion of a dysregulation of the comple-ment system in MS and is in line with previous findings in neurodegenerative diseases. Inter-estingly, and in contrast to NFL, the highest levels were seen in patients with PPMS. Although,this finding should be interpreted with caution due to a small number of patients in the PPMSsubgroup, combining overall progressive patients as a group also displayed higher C3 levels ascompared to the RRMS group. It is likely that increased levels of NFL in RRMS patients are pri-marily related to disease activity driven by adaptive immune responses leading to focal areas ofinflammation and subsequent axonal injury, whereas a shift towards innate immune activationoccurs with progressive disease [6, 41]. Thus, we speculate that increased complement C3, witha potential link to cholinergic activity, is related primarily to more widespread innate inflam-matory activation in the nervous tissue. A related observation is that serum levels of factor H, acomplement regulatory protein, have been reported to be higher in MS patients with a progres-sive disease course [10], even if another study found lower CSF levels of factor H in MS patients[14]. Factor H exerts complement inhibitory functions [42], which may suggest increased con-sumption to balance dysregulated complement activity within the CNS. A previous studyshowed correlation between intrathecal and serum levels of several complement components,for instance C9 and fB [11]. Levels of C9 were found to be lower in MS patients compared tocontrols, perhaps illustrating a different aspect of complement activation than studied here.

Accumulating evidence suggests that CNS inherent complement expression may contributeto neurodegenerative processes occurring after various injuries, including inflammation [43–45]. The complement system is complex, with a large number of proteins that together form anintricate biological network regulated at multiple levels, where many of the components haveboth signalling and direct effector functions [42]. The terminal step of the complement cascadeconsists of the assembly of the membrane attack complex (MAC), composed of the comple-ment proteins C5b-9. However, the initial part of the cascade can be partially activated withoutformation of MAC [46, 47]. Due to this complexity it is difficult to speculate to what degreecomplement activation is an upstream phenomenon contributing to increased neuronal injuryor if increased C3 levels merely reflect a downstream effect of a neurodegenerative process driv-en by other mechanisms. Still, the genetic association between AD risk and the complement re-lated genes clusterin and complement receptor 1 suggests that the complement system can actupstream in the disease process, even if the exact mechanisms remain to be elucidated [48].Also, the association between increased intrathecal C3 in other neurodegenerative diseases [17]and our findings here with the highest CSF C3 levels in patients with more MRI lesions and inthe progressive phases of disease points to potentially important pathways involved in thechronic neurodegenerative phase of MS, which is less understood than its initial relapsing-re-mitting phase. However, it should be noted that the finding of increased C3 in patients with�9cerebral MRI lesions should be interpreted with caution since they were compared with a sub-stantially lower number of cases with little lesion burden.

In this context, the correlation between CSF levels of C3 and BuChE in MS patients is inter-esting. In contrast, even if AChE levels differed between MS patients and controls, there was nosignificant association to C3 at the individual level, arguing against a role for AChE in regula-tion of C3 expression in MS patients. In contrast to our study, Antonelli et al. did not detect asignificant difference in AChE between RRMS and OND [25]. A likely explanation is thechoice of method; we measured AChE activity with an optimal concentration of 0.5–1 mM

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acetylthiocholine (ATCh, an ACh analogue), which has a greater biological relevance. Mea-surements at lower ATCh/ACh concentrations are less informative and might not completelyreflect the full enzymatic capacity of AChE. As mentioned previously a relative increase inBuChE over AChE in the diseased CNS is also supported by previous neuropathological studies[22–24]. Also, recent studies underscore the importance of cholinergic signaling for adaptiveimmune responses in the periphery [49]. There may also be analogous effects mediated byACh in the brain, since both astrocytes and microglia express nicotinic cholinergic receptorsthat attenuate pro-inflammatory responses [50–53]. In addition, a number of studies have re-ported neuroprotective effects mediated through cholinergic signaling across different experi-mental models of neuronal injury [54–56]. However, several questions still remain, such as therelative role of AChE and BuChE for regulating cholinergic tone in CNS tissue, and to what de-gree this balance is important for regulation of inflammation. ACh in biological samples is un-stable and degrades spontaneously without addition of cholinesterase inhibitors, making itunreliable as a biomarker. In addition, the local concentration in the tissue may well be differ-ent from CSF levels.

BuChE is an enzyme with several functions that are still under investigation but not yet fullyunderstood. For instance, BuChE shows peptidase activity and activates trypsin [57], andshows a major role in the metabolic pathways of many substances that reaches the circulation.In addition, the distribution pattern of this enzyme in the CNS is complex and point at impor-tant functions not limited to the classical cholinergic neurotransmission [58]. Thus, we specu-late that BuChE, together with a soluble form of the ACh-synthesizing enzyme ChAT isinvolved in the regulation of extracellular ACh signaling, apart from cholinergic neurotrans-mission, and thereby regulates the functional status of various cholinoceptive, non-excitablecells, such as astrocytes, microglia and oligodendrocytes; all of which should be of high rele-vance to MS in context of inflammatory processes in the CNS [34, 59, 60]. We recently alsodemonstrated that differentiation of neurospheres lead to gliogenesis in synchrony with a re-duction of released ChAT, but with increases in BuChE, i.e. causing a microenvironment favor-ing hypo-cholinergic signaling accompanied with altered cytokine profiles [59, 61].

Adding additional complexity is the possibility of changes in the extra synaptic ChAT as analternative pathway of regulating ACh levels, as recently reported by us [60]. Our findings heresuggest the possibility of a link between BuChE activity and expression of C3, even if it mayalso be that both are regulated by a common upstream event and therefore not directly relatedto each other. One such factor could be reduced physical activity as a result of increasing dis-ability. Somewhat unexpectedly we did not find evidence suggesting that C3 or BuChE are reg-ulated by clinical disease activity in the RRMS group. This further strengthens the notion ofqualitative differences in the inflammatory milieu between RRMS and progressive patients[5–7].

In conclusion we here demonstrate that the levels of C3 in CSF of MS patients correlate todisease course/severity and a biomarker for nerve injury. This finding provides additional sup-port for the notion of complement dysregulation as an integral part of MS disease processes, es-pecially in later disease stages characterized by increased disability and neuronal injury. Thedata also support the notion that innate immune mechanisms are relatively more important inlater stages of the disease [5–7]. We also report the novel observation of a correlation betweenCSF C3 levels and BuChE enzymatic activity, which suggests that expression of complementC3 may be influenced by cholinergic tone. Further studies are needed to address the question ifincreased complement activity reflects an up or down stream event in neurodegenerative pro-cesses occurring in MS and the potential regulatory role of cholinergic signaling.

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Author ContributionsConceived and designed the experiments: SA RL FP. Performed the experiments: SA RL FANSV KS MK. Analyzed the data: SA RL FP MK. Contributed reagents/materials/analysis tools:TO BN KNE TDS FP. Wrote the paper: SA RL FP.

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